Human cytomegalovirus (HCMV), a ubiquitous opportunistic pathogen, is a leading cause of fatal disease in AIDS and organ transplantation patients. This proposal is designed to identify the mechanisms involved in latency and reactivation of HCMV. Monocytes are a persistent site for HCMV infection in the peripheral blood. The low level of virus replication in primary monocyte/macrophage (M/M) cultures has limited the usefulness of this cell system in the past. However, the investigator recently demonstrated, efficient HCMV replication in M/M cultures. Permissiveness in these cells is dependent on stimulation of the monocytes with nonadherent cells and subsequent differentiation into macrophages. This application examines the mechanisms involved in M/M differentiation and the early viral events that regulate HCMV activation. Initially, the investigator will determine whether the block in HCMV replication in unstimulated monocytes is at the transcriptional level by performing run-on and RT-PCR analysis. He also will determine whether individual or combinations of protein products of the major immediate early transcriptional units (MIE proteins) introduced into monocytes by lipofection of cDNA constructs or by microinjection of purified protein can override the block in viral replication in unstimulated cells. In addition, the expression of MIE products in HCMV-infected, differentiated M/M cultures will be examined by northern analysis, RT- PCR, and western blot to identify the predominant protein isoforms. New isoforms, if present, will be cloned and tested for their ability to activate or repress immediate early (IE), early (E), and late (L) promoters by using M/M transfection assays. MIE isoforms absent in M/M will be introduced to infected M/M to examine their roles in activation or repression of HCMV expression. In addition, mechanisms involved in MIE protein transactivation and repression of various promoters will be examined in vitro with purified recombinant proteins. In these experiments, the interactions of IE72 and IE86 with TBP and of IE86 binding to the CRS element will be examined by gel mobility shift experiments.